Team:UI-Indonesia/Basic Part
Parts
Enhanced green fluorescent protein (EGFP) coding sequence (human-optimized)
BBa_K2050420
TThe green fluorescent protein (GFP) is a protein originally obtained from the jellyfish Aequorea victoria that emits green fluorescence upon exposure to light in the blue to ultraviolet range and is typically used in molecular biology and genetics as a reporter. The enhanced GFP (or EGFP) was developed to allow for its practical usage in mammalian cells. Under our lab-developed promoter, MAX promoter, we have succesfully validated our part BBa_K2050420 EGFP. We tested this on E. coli TOP-10 within 18 hours of incubation in 37 degree celcius incubation. The same plasmid which carried different insert was used as a control.
Figure 1. Eschericia coli containing MAX promoter coupled with Bba_K2050420 on UV light exposure
This EGFP which was optimized for mammalian cells expression was unable to be characterized in mammalian cells because there was a problem in assembling our plasmid from the g-blocks. Nevertheless, we are assured that this EGFP should work well in mammalian cells because the MAX promoter was an optimized mammalian promoter which should work well in mammalian cell.
Thosea Asigna Virus 2A peptide linker (T2A)
BBa_K2050421
2A peptide is a self-cleaving peptide linker. The sequence encoding the 2A peptide is typically included between the coding sequences of two proteins without stop codons to co-express the two proteins in a single vector. The two coding sequences will be transcribed onto one mRNA molecule along with the coding sequence for the 2A peptide. During translation, however, the peptide will cleave the peptide bond between the glycine and the proline residue on its C-terminus. The result is two different polypeptide chains.
The coding sequence for 2A peptide is far shorter than that of an IRES, making it preferable in constructing a multicistronic vector. The consequence of using the 2A peptide linker, however, is that the upstream protein will have additional residues from the 2A peptide on its C-terminus and the downstream protein will start with a proline
Mouse H1 promoter (RNA Polymerase III promoter)
BBa_K2050422
RNA polymerase III is responsible for transcribing small RNA molecules including the 5S rRNA and tRNAs. RNA polymerase III promoters, including the H1 promoter, is useful in creating synthetic functional RNA-encoding genes. RNA polymerase III promoters are considerably smaller than RNA polymerase II promoters and initiates expression at ten times the rate RNA polymerase II promoters do. Examples on using functional RNA genes include miRNA expression, guide RNA expression for CRISPR technology, and immunostimulatory RNA (isRNA) expression, the latter used in many projects (including ours) on vaccination.
The use of RNA molecules as a vaccine adjuvant hinges on the interaction of those RNA molecules with the retinoic acid-inducible gene 1 (RIG-1) receptor, which is a pathogen-associated molecular pattern recognition receptor specialized in detecting non-self RNA molecules and triggering the innate immunity upon recognizing such molecules. The presence of 5'-PPP on these molecules distinguish them from self RNA molecules, in which the 5'-PPP is usually moved or masked. Because RNA polymerase III transcription does not initiate 5-capping like RNA polymerase II transcription does, the use of RNA polymerase III promoters have this as an additional advantage for isRNA expression. The use of a mouse H1 promoter instead of that of human is preferable for human applications as it limits the probability for homologous recombination with the human genome.
tRNA(Val) and linker
BBa_K2050423
The tRNA(Val) and linker can be used to facilitate the export of a functional non-coding RNA into the cytosol (using the inherent tRNA export mechanism).
Adenoviral VA1 RNA gene
BBa_K2050424
The VA1 RNA gene is an RNA polymerase III-driven gene that encodes the viral RNA VA1. By its nature, the RNA molecule facilitates infection by inhibiting the protein kinase R response, which is activated in infected cells and acts to globally suppress gene expression to inhibit viral replication. Co-expressing the VA1 RNA gene with a desired non-coding RNA gene allows for the expression of the desired RNA molecule, which would generally trigger PKR response, without sacrificing RNA polymerase II transcription.
Figure 2. Agarose gel electrophoresis on 1,3% agarose, 100V/37 minutes.
Our parts were working as expected by showing on agarose gel electrophoresis, except for BBa_K2050421 (Peptide 2A Linker). Because of this reasons, we cancelled sending this parts to the igem HQ.
